CN101732706A - Method for preparing living paratyphoid vaccine for piglets and product thereof - Google Patents
Method for preparing living paratyphoid vaccine for piglets and product thereof Download PDFInfo
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Abstract
The invention discloses a method for producing living paratyphoid vaccine for piglets and a product thereof. The method comprises the following steps: (1) preparing primary strains; (2) preparing secondary strains; and (3) culturing bacterial liquid: inoculating the secondary strains into a culture medium for culturing, after culturing, immediately adding a stabilizing agent which is sterilized and preheated to 37 DEG C to ensure that the vaccine contains 1.5 percent of gelatin and 5 percent of sucrose, and mixing uniformly to obtain the bacterial liquid, wherein the culture medium in the step (3) consists of a common broth medium and a synthetic medium in the volume ratio of 2:1. In the living paratyphoid vaccine for piglets produced by the method, the cultured bacteria count is over 50 billion per milliliter; the maximum cultured bacteria count is 58 billion per milliliter; and the freeze-drying death rate is reduced to 20-40 percent. The method for producing the living paratyphoid vaccine for the piglets has the advantages of high bacterial count of the living vaccine, low freeze-drying death rate, easy production, high yield, low production cost, and the like.
Description
Technical field
The present invention relates to a kind of preparation method of animal live vaccine, relate in particular to a kind of preparation method of living paratyphoid vaccine for piglets and the product for preparing by this method, belong to the living paratyphoid vaccine field.
Background technology
Baby swine paratyphoid is acute, the subacute or chronic infectious disease that is caused by Salmonella choleraesuls.Mainly betide 2-4 monthly age piglet, hazardness is very big, acute case mainly shows as septicemia, symptom is body temperature rising suddenly, lassitude, does not eat, have between the later stage that skin has the aubergine speckle under dysentery, dyspnea, the basal part of the ear, front and the abdomen, sometimes 24h is interior dead after symptom occurring, and case fatality rate is very high.Subacute and chronic sympton shows as and becomes thin, dysentery and enteritis, and the state of an illness is often delayed 2-3 week or longer, and last skeletonize dies of exhaustion.Soviet citizen Yi Wanuofuyin successfully developed the baby swine paratyphoid inactivated vaccine in 1948, had obtained Stalin's medal.
Use inactivated vaccine abroad very early; China also brings into use inactivated vaccine in five sixties; but the result of use of inactivated vaccine is very undesirable; nineteen sixty China once made contain various different adjuvants inactivated vaccine totally 52 batches 235 piglets are carried out immunity test; all immunity twice, protective rate only is 29.9% behind the counteracting toxic substances.China in 1961 begin that less-virulent strain is cultivated and the development of weak toadstool Seedling, selected a strain virulence in 1964 a little less than and stable, the good strain of immunogenicity, be numbered C500.
A little less than producing C500, in the toadstool Seedling process, produce vaccine, have the shortcoming that labor intensity is big and yield poorly, begin each biological factory from early seventies and use the production of liquid ventilation culture method successively instead because of adopting big flat bottle agar to cultivate.But because aerobic culture has part thalline life comparatively fragile, the vaccine lyophilizing appears easily and after the viable bacteria rate lower, and cause that the piglet reaction is bigger, and occur unsafe situation once in a while.After each producer improves cultivation and freeze drying technology and carries out the vaccine oral test, the effect of oral test proof oral immunity and the effect of injection are good equally, and oral viable bacteria rate to be low to moderate 37% vaccine also very safe, in view of the above the result stipulated lyophilizing viable bacteria rate be not less than 50% be used for injection or oral; Lyophilizing viable bacteria rate is lower than 50% vaccine and can not injects but can be used for oral.This regulation can guarantee the use of dispatching from the factory of more vaccine.In " veterinary biologics rules " were listed liquid ventilation culture method approval in 1976 by China, and many biological product manufacturer puts into production in the whole nation, applies.The method is leveraged to the present always, and this cultural method exists the problem that the bacterium number is low, the lyophilizing mortality rate is high always, and the production difficulty is big, yields poorly, the cost height.Because of the huge market demand, the phenomenon that each manufacturer all occurs supply shortage often takes place, and is perplexing each manufacturer for many years always.
" veterinary biologics rules " have carried out concrete standard to the production method of living paratyphoid vaccine for piglets, and are as follows by " rules " production method:
1, the preparation of first class inoculum:
Strain is with the ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, it is individual for circle, neat in edge, projection, translucent, slightly moistening smooth type median size bacterium colony 5-10 to choose bacterium colony, some of combined inoculations in the plain agar inclined-plane, cultivate 24h through pure check for 37 ℃, and do the slide agglutination test passed examination with 1/500 acriflavinium chloride solution, as first class inoculum, 2-8 ℃ of preservation, should be no more than 2 months transplantation 1-2 time around here, subculture on culture medium was no more than for 5 generations.
2, the preparation of second class inoculum:
Get the flat bottle of first class inoculum inoculation ordinary broth agar, cultivate 24h (the plain agar culture washes with ordinary broth and makes the suitable bacterium liquid of concentration), after the pure passed examination, promptly can be used as seed for 37 ℃.2-8 ℃ of preservation, should be no more than 2.
3, bacterium liquid is cultivated: the 1%-2% by the culture medium total amount is inoculated in the ordinary broth that contains the 1%-1.5% peptone with second class inoculum, at 37 ℃ of aerobic culture 18-21h, add appropriate amount of defoamer in the incubation as required, and add an amount of sterilization 40% Glucose Liquid with control PH according to PH rising situation.Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5% gelatin and 5% sucrose, a fully part quantitatively packing in accordance with regulations behind the mixing.Every part viable bacteria is no less than 3,000,000,000.
Produce according to the method described above, cultivate the bacterium number generally at 180-260 hundred million/ml, join Seedling by 3,000,000,000 calculating of every part viable bacteria, maximum branch loading amount is the 3.8ml/ bottle, and the rules regulation is minimum to be 20 part/bottles, this bacterium lyophilizing mortality rate is at 40%-60%, calculate part standard of stipulating near " rules " of bowing most by the highest cultivation bacterium several 26,000,000,000 and 60% mortality rate, this cultural method exists the problem that the bacterium number is low, the lyophilizing mortality rate is high always, and the production difficulty is big, yield poorly, the cost height, have much room for improvement.
Summary of the invention
Technical problem to be solved by this invention is low, the lyophilizing mortality rate height of the existing bacterium number of production method that overcomes existing living paratyphoid vaccine for piglets, the production difficulty is big, yield poorly, problems such as cost height, a kind of production method of new living paratyphoid vaccine for piglets is provided, this production method can make the antibacterial culturing time shorten, and cultivates the bacterium number and obviously improves, and the lyophilizing mortality rate descends significantly.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of production method of living paratyphoid vaccine for piglets, comprise: the preparation of (1) first class inoculum: the Salmonella choleraesuls strain is with the ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, choose median size bacterium colony 5-10, combined inoculation is cultivated 24h for 37 ℃, after the assay was approved as first class inoculum in the plain agar inclined-plane; (2) preparation of second class inoculum: get the flat bottle of first class inoculum inoculation ordinary broth agar, cultivate 24h for 37 ℃, after the passed examination, as secondary seed; (3) bacterium liquid is cultivated: second class inoculum is inoculated in the culture medium cultivates; Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5% gelatin and 5% sucrose, fully mixing; Wherein, the culture medium described in the step (3) is made up of according to 2: 1 volume ratio broth medium and synthetic medium; Described broth medium consists of: beef: 500g, peptone: 10-15g, sodium chloride: 5g, distilled water or deionized water: 1000ml; Consisting of of described synthetic medium: beef extract powder: 10g, peptone: 10g, yeast powder: 10g, glucose: 2g, sodium chloride: 5g; Add water to 1000ml.The present invention also by a large amount of experiment discoveries, adopts following means in aforementioned production method, for shortening the antibacterial culturing time, improving methods such as cultivating bacterium number and reduction lyophilizing mortality rate all has obvious effects:
Get first class inoculum inoculation plain agar or ordinary broth in the step (2), 37 ℃ are cultivated behind the 24h with inserting loading amount behind the 40% sterilization glucose solution flushing lawn is among 10,000 bottles of the 3000ml, 37 ℃ of cultivation 20-22h; After the passed examination, be re-used as secondary seed;
Inoculum concentration described in the step (3) is preferably: 3% inoculum concentration by the culture medium gross weight is inoculated into secondary seed in the culture medium; Described cultivation is preferably aerobic culture 15h under 37 ℃ of conditions, and is wherein preferred, strengthens aerobic culture 15h gradually under 37 ℃ of temperature;
The present invention is directed to low, the lyophilizing mortality rate height of the existing bacterium number of living paratyphoid vaccine for piglets production method, the production difficulty is big, yield poorly, problems such as cost height, carried out the research that improves the living paratyphoid vaccine for piglets preparation method targetedly, done a large amount of explorations, development test from production technology and culture medium prescription aspect.The living paratyphoid vaccine for piglets that the inventive method is produced is cultivated the bacterium number and is broken through 50,000,000,000/ml, is up to 58,000,000,000/ml, and the lyophilizing mortality rate is reduced to 20-40%.Be 20 part/bottles, 30 part/bottles, 40 part/bottles to the living paratyphoid vaccine for piglets specification in " veterinary biologics rules "; According to 15 batches the live vaccine that the inventive method is produced, wherein 11 batches reach 30 part/bottles, and 4 batches reach 40 part/bottles.The inventive method has that the live vaccine bacterium that is produced is counted height, the lyophilizing mortality rate is low, produces output height, low cost and other advantages easily.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
One, the preparation of ordinary broth
Beef: 500g
Distilled water or deionized water: 1000ml
Peptone: 10-15g
Sodium chloride: 5g;
Above-mentioned each component is dissolved in distilled water or the deionized water, autoclave sterilization, promptly.
Two, the preparation of synthetic medium
Beef extract powder: 10g, peptone: 10g, yeast powder: 10g, glucose: 2g, sodium chloride: 5g; Add water to 1000ml, autoclave sterilization, promptly.
Embodiment 1
1, the preparation of first class inoculum:
With Salmonella choleraesuls strain (purchase) in China Veterinary Drugs Supervisory Inst. with ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, choose bacterium colony for circular, neat in edge, projection, translucent, slightly moistening smooth type median size bacterium colony 5-10, some of combined inoculations in the plain agar inclined-plane, cultivate 24h through pure check for 37 ℃, and do the slide agglutination test passed examination with 1/500 acriflavinium chloride solution, as first class inoculum, 2-8 ℃ of preservation, should be no more than 2 months transplantation 1-2 time around here, subculture on culture medium was no more than for 5 generations.
2, the preparation of second class inoculum:
Hook up the flat bottle of first class inoculum streak inoculation ordinary broth agar, 37 ℃ cultivate 24 after, be among 10,000 bottles of the 3000ml with inserting loading amount behind the 40% sterilization glucose solution flushing lawn, cultivate 20-22h for 37 ℃, after the passed examination, promptly can be used as secondary seed purely.2-8 ℃ of preservation, should be no more than 3.
3, bacterium liquid is cultivated:
Second class inoculum is inoculated in two parts of ordinary broths that contain the 1%-1.5% peptone and a synthetic medium (2: 1) by 3% of culture medium total amount, with 37 ℃ of aerobic culture 15h of intelligent microorganism fermentation tank, will adopt in the incubation and strengthen ventilation gradually and cultivate, and to add an amount of sterilization 40% Glucose Liquid according to pH rising situation be 7.4-7.6 with the control pH value.Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5% gelatin and 5% sucrose, a fully part quantitatively packing in accordance with regulations behind the mixing.Every part viable bacteria reaches 4,000,000,000.
Embodiment 2
1, the preparation of first class inoculum:
With Salmonella choleraesuls strain (commerce that please provide the Salmonella choleraesuls strain is bought approach) with the ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, choose bacterium colony for circular, neat in edge, projection, translucent, slightly moistening smooth type median size bacterium colony 5-10, some of combined inoculations in the plain agar inclined-plane, cultivate 24h through pure check for 37 ℃, and do the slide agglutination test passed examination with 1/500 acriflavinium chloride solution, as first class inoculum, 2-8 ℃ of preservation, should be no more than 2 months transplantation 1-2 time around here, subculture on culture medium was no more than for 5 generations.
2, the preparation of second class inoculum:
Hook up the flat bottle of first class inoculum streak inoculation ordinary broth agar, after 37 ℃ of cultivations 24, after the pure passed examination, promptly can be used as secondary seed.2-8 ℃ of preservation, should be no more than 3.
3, bacterium liquid is cultivated:
Second class inoculum is inoculated in two parts of ordinary broths that contain the 1%-1.5% peptone and a synthetic medium (2: 1) by 3% of culture medium total amount, with 37 ℃ of aerobic culture 15h of intelligent microorganism fermentation tank, adopt in the incubation and strengthen ventilation gradually and cultivate, and to add an amount of sterilization 40% Glucose Liquid according to pH rising situation be 7.4-7.6 with the control pH value; Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5% gelatin and 5% sucrose, a fully part quantitatively packing in accordance with regulations behind the mixing.Every part viable bacteria is no less than 3,000,000,000.
Embodiment 3
1, the preparation of first class inoculum:
With Salmonella choleraesuls strain (commerce that please provide the Salmonella choleraesuls strain is bought approach) with the ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, choose bacterium colony for circular, neat in edge, projection, translucent, slightly moistening smooth type median size bacterium colony 5-10, some of combined inoculations in the plain agar inclined-plane, cultivate 24h through pure check for 37 ℃, and do the slide agglutination test passed examination with 1/500 acriflavinium chloride solution, as first class inoculum, 2-8 ℃ of preservation, should be no more than 2 months transplantation 1-2 time around here, subculture on culture medium was no more than for 5 generations.
2, the preparation of second class inoculum:
Hook up the flat bottle of first class inoculum streak inoculation ordinary broth agar, 37 ℃ cultivate 24 after, be among 10,000 bottles of the 3000ml with inserting loading amount behind the 40% sterilization glucose solution flushing lawn, cultivate 20-22h for 37 ℃, after the passed examination, promptly can be used as secondary seed purely.2-8 ℃ of preservation, should be no more than 3.
3, bacterium liquid is cultivated:
Second class inoculum is inoculated in two parts of ordinary broths that contain the 1%-1.5% peptone and a synthetic medium (2: 1) by 3% of culture medium total amount, with 37 ℃ of aerobic culture 20h of intelligent microorganism fermentation tank, and to add an amount of sterilization 40% Glucose Liquid according to pH rising situation be 7.4-7.6 with the control pH value.Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5% gelatin and 5% sucrose, a fully part quantitatively packing in accordance with regulations behind the mixing.Every part viable bacteria is no less than 3,000,000,000.
Test example 1
One, test material
For trying vaccine: the live vaccine that embodiment 1-3 is prepared; Be divided into and be 15 batches;
Control vaccine: the living paratyphoid vaccine for piglets of being produced according to " veterinary biologics rules "; Be divided into 9 batches;
Two, test method and result
Adopt identical freeze-drying curve for the examination vaccine with control vaccine, freeze-drying curve is-15 ℃ of cartonnings, pre-freeze-40 ℃ 4h, sublimation drying-2 ℃ 25h, 32 ℃ of 7h of adsorption stripping and dry.The count of bacteria culture medium is ordinary broth agar (ordinary broth adds 1.5% agar), adopts " veterinary biologics rules " viable bacteria counting method, sees Table 1 for cultivation bacterium number that tries vaccine and control vaccine and the contrast of lyophilizing mortality rate.
Table 1 is for the cultivation bacterium number and the lyophilizing mortality rate contrast table of examination vaccine and control vaccine
From result of the test as seen, adopt the cultivation bacterium number of the prepared live vaccine of production method of the present invention to break through 50,000,000,000/ml, be up to 58,000,000,000/ml, the lyophilizing mortality rate is reduced to 20-40%; " veterinary biologics rules " specification is 20 part/bottles, 30 part/bottles, 40 part/bottles; Adopt the inventive method to produce 15 batches of living paratyphoid vaccine for piglets altogether, wherein 11 batches reach 30 part/bottles, and 4 batches reach 40 part/bottles.
Claims (9)
1. the production method of a living paratyphoid vaccine for piglets, comprise: the preparation of (1) first class inoculum: with the Salmonella choleraesuls strain with ordinary broth dilution or after ordinary broth is cultivated breeding, inoculation plain agar flat board, cultivate 18-20h for 37 ℃, choose bacterium colony 5-10, combined inoculation is cultivated 24h for 37 ℃, after the assay was approved as first class inoculum in the plain agar inclined-plane; (2) preparation of second class inoculum: get the flat bottle of first class inoculum inoculation ordinary broth agar, 37 ℃ are cultivated behind the 24h with access loading amount behind the 40% sterilization glucose solution flushing lawn is that 37 ℃ of cultivations are after the passed examination, as secondary seed among 10,000 bottles of the 3000ml; (3) bacterium liquid is cultivated: second class inoculum is inoculated in the culture medium cultivates; Add immediately through sterilizing and being preheated to 37 ℃ stabilizing agent after cultivating end, make vaccine contain 1.5wt% gelatin and 5wt% sucrose, fully mixing; It is characterized in that: the culture medium described in the step (3) is made up of according to 2: 1 volume ratio broth medium and synthetic medium.
2. according to the described production method of claim 1, it is characterized in that: described broth medium consists of: beef 500g, peptone 10-15g, sodium chloride 5g, distilled water or deionized water 1000ml; Consisting of of described synthetic medium: beef extract powder: 10g, peptone: 10g, yeast powder: 10g, glucose: 2g, sodium chloride: 5g; Add water to 1000ml.
3. according to the described production method of claim 1, it is characterized in that: get the flat bottle of first class inoculum inoculation ordinary broth agar in the step (2), 37 ℃ are cultivated behind the 24h with inserting loading amount behind the 40% sterilization glucose solution flushing lawn is among 10,000 bottles of the 3000ml, 37 ℃ of cultivation 20-22h; After the passed examination, be re-used as secondary seed.
4. according to the described production method of claim 1, it is characterized in that: 3% inoculum concentration by the culture medium gross weight in the step (3) is inoculated into secondary seed in the culture medium.
5. according to the described production method of claim 1, it is characterized in that: described cultivation is aerobic culture 15h under 37 ℃ of conditions.
6. according to the described production method of claim 1, it is characterized in that: described cultivation is to strengthen aerobic culture 15h gradually under 37 ℃ of temperature.
7. any one production method of claim 1-6 is produced the live vaccine that obtains.
8. the purposes of the described live vaccine of claim 7 in preparation prevention or treatment baby swine paratyphoid medicine.
9. the pharmaceutical composition of prevention or treatment baby swine paratyphoid is characterized in that: be made up of the described live vaccine of claim 7 and pharmaceutically acceptable carrier or excipient.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
| CN111849834A (en) * | 2020-08-07 | 2020-10-30 | 甘肃省畜牧兽医研究所 | Live vaccine culture medium and application thereof, and yak paratyphoid live vaccine and preparation method thereof |
| CN114134097A (en) * | 2021-12-09 | 2022-03-04 | 国药集团扬州威克生物工程有限公司 | Piglet paratyphoid attenuated strain fermentation culture, live vaccine and preparation method thereof |
| CN115058360A (en) * | 2022-06-16 | 2022-09-16 | 重庆澳龙生物制品有限公司 | Preparation method of salmonella equine abortion live vaccine |
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| GB1085956A (en) * | 1963-09-10 | 1967-10-04 | Nat Res Dev | Improvements relating to vaccines |
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| 张金平 等: "仔猪副伤寒（C500株）培养菌数比较试验", 《中国畜牧兽医学会生物制品学分会第十次学术研讨会论文集》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
| CN101912608B (en) * | 2010-08-17 | 2012-08-22 | 北京中海生物科技有限公司 | Method for producing paratyphus living vaccine for piglets |
| CN111849834A (en) * | 2020-08-07 | 2020-10-30 | 甘肃省畜牧兽医研究所 | Live vaccine culture medium and application thereof, and yak paratyphoid live vaccine and preparation method thereof |
| CN114134097A (en) * | 2021-12-09 | 2022-03-04 | 国药集团扬州威克生物工程有限公司 | Piglet paratyphoid attenuated strain fermentation culture, live vaccine and preparation method thereof |
| CN115058360A (en) * | 2022-06-16 | 2022-09-16 | 重庆澳龙生物制品有限公司 | Preparation method of salmonella equine abortion live vaccine |
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